1. Field of the Invention
The present invention relates to a polyhydroxyalkanoate (PHA) as a new type of polyester. The present invention also relates to a method of producing the PHA using a microorganism capable of producing the PHA and accumulating the PHA in its body. The present invention also relates to a compound suitably used for the culture of the microorganism.
2. Related Background Art
Hitherto, many microorganisms have been reported to produce poly-3-hydroxy butyric acid (PHB) and other PHAs and accumulate the same in their bodies (xe2x80x9cBiodegradable Plastic Handbookxe2x80x9d, edited by Biodegradable Plastic Society, NTS Co. Ltd., pp. 178-197 (1995)). These polymers may be used for production of a variety of products using melt processing and the like as with conventional polymers. Furthermore, they have an advantage that they are fully decomposed by microorganisms in the natural world because they are biodegradable, and therefore there are no possibilities that they remain in the natural environment to cause contamination as is the case with many conventional synthetic polymer compounds. Those biodegradable polymer compounds have excellent biocompatibility, and their application as medical soft materials and the like are expected.
It is known that such microorganism-produced PHA may have a variety of compositions and structures depending on the type, culture composition, culture condition and the like of the microorganism for use in the production of the PHA, and studies as to control of such compositions and structures have been conducted mainly in terms of improvement of properties of the PHA.
[1] First, biosynthesized PHAs obtained by polymerizing monomer units having a relatively simple structure such as 3-hydroxy butyric acid (hereinafter abbreviated as 3HB) include:
(a) PHA containing 3HB and 3-hydroxy valeric acid (hereinafter referred to as 3HV).
Japanese Patent Publication No. 6-15604, Japanese Patent Publication No. 7-14352, Japanese Patent Publication No. 8-19227 and Japanese Patent Application Laid-Open No. 5-7492.
(b) PHA containing 3HB and 3-hydroxy hexanoic acid (hereinafter referred to as 3HHx).
Japanese Patent Application Laid-Open No. 5-93049 and Japanese Patent Application Laid-Open No. 7-265065.
(c) PHA containing 3HB and 4-hydroxy butyric acid (hereinafter referred to as 4HB).
Japanese Patent Application Laid-Open No. 9-191893.
(d) PHA containing 3-hydroxy alkanoate having 6 to 12 carbon atoms.
Japanese Patent No. 2642937.
(e) PHA biosynthesized using a single fatty acid as a carbon source. The product is almost the same as (d).
Appl. Environ. Microbiol, 58 (2), 746 (1992).
They are all PHA composed of monomer units each having an alkyl group in the side chain, synthesized through xcex2-oxidation of hydrocarbon by a microorganism or synthesis of fatty acid from a sugar, namely xe2x80x9cusual PHAxe2x80x9d.
[2] However, when considering a wider range of application of such microorganism-produced PHA, for example application as a functional polymer, it is expected that PHA having a substituent group other than an alkyl group introduced into the side chain, namely xe2x80x9cunusual PHAxe2x80x9d will be very useful. Examples of the substituent group include groups containing an aromatic ring (phenyl group, phenoxy group, etc.), unsaturated hydrocarbons, ester group, allyl group, cyano group, halogenated hydrocarbons and epoxide. Among them, in particular, PHA having an aromatic ring have been intensively studied.
(a) PHA having a phenyl group or a partially substituted phenyl group.
It is reported in Macromol. Chem., 191, 1957-1965 (1990) and Macromolecules, 24, 5256-5260 (1991) that using 5-phenyl valeric acid as a matrix, Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-phenyl valeric acid as a unit.
It is reported in Macromolecules, 29, 1762-1766 (1996) that using 5-(4xe2x80x2-tolyl) valeric acid as a matrix, Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-(4xe2x80x2-tolyl) valeric acid as a unit.
It is reported in Macromolecules, 32, 2889-2895 (1999) that using 5-(2xe2x80x2, 4xe2x80x2-dinitrophenyl) valeric acid as a matrix, Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-(2xe2x80x2, 4xe2x80x2-dinitrophenyl) valeric acid and 3-hydroxy-5-(4xe2x80x2-nitrophenyl) valeric acid as units.
(b) PHA containing a phenoxy group or a partially substituted phenoxy group.
It is reported in Macromol. Chem. Phys., 195, 1665-1672 (1994) that using 11-phenoxy undecylic acid as a matrix, Pseudomonas oleovorans produces a PHA copolymer of 3-hydroxy-5-phenoxy valeric acid and 3-hydroxy-9-phenoxy nonanoic acid.
In Japanese Patent No. 2989175 is disclosed an invention relating to a homopolymer composed of 3-hydroxy-5-(monofluorophenoxy) pentanoate (3H5-(MFP)P) units or 3-hydroxy-5-(difluorophenoxy) pentanoate (3H5(DFP)P) units, and a copolymer containing at least 3H5-(MFP)P units or 3H5(DFP)P units; Pseudomonas putida synthesizing these polymers; and a method of producing the above described polymers using a Pseudomonas, and it is described that as an effect thereof, a long-chain fatty acid having a substituent group can be assimilated to synthesize a polymer having a phenoxy group with the end of the side chain substituted with one or two fluorine atoms, and stereoregularity and water repellency can be given while maintaining a high melting point and good processability.
Studies on substances substituted with a cyano group and a nitro group have been conducted in addition to such a substance substituted with a fluorine group.
It is reported in Can. J. Microbiol., 41, 32-43 (1995) and Polymer International, 39, 205-213 (1996) that Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442 strains are used to produce PHA containing 3-hydroxy-p-cyanophenoxy hexanoic acid or 3-hydroxy-p-nitrophenoxy hexanoic acid as a monomer unit using octanoic acid and p-cyanopenoxy hexanoic acid or p-nitrophenoxy hexanoic acid as a matrix.
These reported PHAs each have an aromatic ring as the side chain unlike general PHA having an alkyl group as the side chain, and are useful in obtaining a polymer having properties originating from the PHA.
[3] In addition, as a new category, studies are conducted aimed at producing PHA having a suitable functional group in the side chain, and using the functional group to produce a new function, beyond just changing the properties.
It is reported that Macromolecules, 31, 1480-1486 (1996) and Journal of Polymer Science: Part A: Polymer Chemistry, 36, 2381-2387 (1998) that PHA containing a unit having a vinyl group at the end of the side chain was synthesized, and was thereafter epoxidized with an oxidizing agent, whereby PHA containing an epoxy group having a high reactivity at the end of the side chain could be synthesized.
Also, as an example of synthesis of PHA containing a unit having thioether expected to have a high reactivity, it is reported in Macromolecules, 32, 8315-8318 (1999) that using 11-(phenylsulfanyl)valeric acid as a matrix, Pseudomonas putida 27N01 strain produces a PHA copolymer of 3-hydroxy-5-(phenylsulfanyl)valeric acid and 3-hydroxy-7-(phenylsulfanyl)heptanoic acid.
As for the PHA containing a 3-hydroxy-(phenylsulfanyl)alkanoic acid unit having an S atom in the side chain, of the above PHAs, more intensive studies will be expectedly conducted in developing functional PHA in the future in terms of its high reactivity. However, not so many studies have been conducted on this type of PHA, and the reported cases relating to such PHA are only the above described cases. Also, for developing functional PHA by chemically modifying the side chain of obtained PHA, it is desired that the PHA has a side chain having a higher reactivity than a phenyl group, such as for example a thienyl group, but any case of production of such PHA has not yet been reported.
In view of those problems, an object of the present invention is to provide a novel PHA and a method of producing the same.
The inventors have intensively conducted studies to solve the above-described problems and accomplished the following invention. Specifically, a first aspect of the present invention is polyhydroxyalkanoate itself characterized by having a unit represented by Chemical Formula [1]: 
wherein n denotes an integer of 1 to 9.
Further, the polyhydroxyalkanoate of the present invention is a polyhydroxyalkanoate characterized in that a unit other than the unit represented by Chemical Formula [1] comprises at least one of units represented by Chemical Formula [2]: 
wherein m denotes an integer of 0 to 8; and Chemical Formula [12]: 
wherein 1 denotes 3 or 5.
The polyhydroxyalkanoate obtained by the present invention is a polyhydroxyalkanoate having a number average molecular weight within the range of 10,000 to 1,000,000.
Also, the polyhydroxyalkanoate obtained by the present invention has asymmetric carbon atoms as shown in Chemical Formulas [1], [2] and [12], which accounts for a large number of potential stereoisomers.
However, the production method according to the present invention is characterized in that a microorganism is used to produce polyhydroxyalkanoate, and thus the 3-hydroxyalkanoic acid monomer units are all in R-configuration.
A second aspect of the present invention is a method of producing the above described polyhydroxyalkanoate, characterized in that a microorganism is cultured in a medium containing a compound represented by Chemical Formula [3]: 
wherein k denotes an integer of 3 to 11.
More particularly, it is a method of producing a polyhydroxyalkanoate, characterized in that a microorganism is cultured in a medium containing 5-(2-thienylsulfanyl)valeric acid represented by Chemical Formula [4]: 
or 6-(2-thienylsulfanyl)hexanoic acid represented by Chemical Formula [5]: 
A third aspect of the present invention is xcfx89-(2-thienylsulfanyl)alkanoic acid itself represented by Chemical Formula [11]: 
wherein k denotes an integer of 4 to 11, which is required for production of the polyhydroxyalkanoate of the present invention.
The method of producing xcfx89-(2-thienylsulfanyl)alkanoic acid used in the present invention and represented by Chemical Formula [11]: 
wherein k denotes an integer of 4 to 11, comprises the step of reacting any one of a bromoalkanoic acid, a bromoalkanoic acid ester (referred to as xe2x80x9cbromoalkanoatexe2x80x9d) derivative, a bromoalkanol, a dibromoalkane and a lactone with thiophene-2-thiol.